Degradation and detection of TSE infectivity

ABSTRACT

A transmissible spongiform encephalopathy (TSE) agent is inactivated by exposing the TSE agent to a thermostable proteolytic enzyme at elevated temperature and at acid or alkaline pH. Following this step, or separately, presence of TSE infectivity is detected by detection of dimers of prion protein.

[0001] The invention is in the field of methods and compositions for thesterilisation of materials and apparatus that may have been contaminatedwith infectious agents and for detection of those agents. In particular,the invention relates to methods for the inactivation and detection oftransmissible spongiform encephalopathy (TSE) agents and providescompositions for degrading and detecting TSE located on or withininfected materials.

[0002] Transmissible spongiform encephalopathies (TSEs) are a group offatal neurological diseases that include Creutzfeld-Jacob disease (CJD)and Kuru in humans, bovine spongiform encephalopathy (BSE) in cattle andScrapie in sheep. TSEs are characterised by the conversion of a normalhost protein into a pathogenic protein within the brain tissue of aninfected animal. The pathogenic form of protein is often referred to asa prion and is highly resistant to physical and chemical degradation.The prion is believed to be the transmissive agent through which the TSEdisease is passed on between animals.

[0003] There has been considerable public alarm in recent years over therisks associated with consumption of meat products, and especially beef,potentially infected with BSE, the bovine form of TSE. Much of thisconcern is associated with the belief that the BSE prion when eaten by ahuman may in some cases cause the incurable human form of the disease,referred to as variant CJD (vCJD). Rigorous practices have been adoptedin the agricultural and meat rendering industries to reduce the risk ofcross contamination between BSE infected carcasses and meat that isdestined for human consumption or other animal derived products such astallow. However, BSE infected animals can still be unintentionallyprocessed in abattoirs, especially if the animal is in the early stagesof the disease and therefore undetected as an infected TSE host. Thereis also a considerable risk in disposal of known BSE infected material,particularly if the equipment used in the disposal operation is thenreused in normal rendering practices without adequate sterilisation.

[0004] Sterilisation of instruments and equipment following potentialexposure to TSE infected tissue is of primary importance. In particular,surgical equipment such as scalpels, forceps and endoscopes should bethoroughly sterilised before use on patients to avoid diseasetransmission.

[0005] It has been reported that the CJD infectious agent wasaccidentally transferred on surgical electrodes, inserted into the brainof a human patient with CJD, to two other previously uninfected patients(Bernouli et al (1977) The Lancet i: pp478-479). The electrodesconcerned were sterilised with ethanol and formaldehyde vapour betweeneach procedure, conditions previously thought to be sufficient toeliminate virtually all infectious agents, and yet the CJD infectiousagent was able to withstand such harsh conditions and infect therecipient patients' brain tissue.

[0006] TSE transmission is typically observed in cases where infectedmaterial is transferred between animals or implanted into an animal. Asdescribed previously, incomplete or inadequate sterilisation of surgicalinstruments can lead to such transfer of infected material betweenpatients. Even the most rigorous chemical cleaning and steamsterilisation procedures can fail to remove blood and tissue fromsurgical instruments, especially in the jaws or joints of forceps andclamps (Laurenson (1999) The Lancet, 20 November). Thus, the risk ofunintentional TSE transfer can be unnecessarily high.

[0007] TSE agents, or prions, are known to be highly resistant todenaturation and degradation, more so than would normally be expectedfor a protein. Taylor (J. Hosp. Infect. (1999) 43 supplement, pp S69-76)reviews a number of methods for inactivating prion proteins.

[0008] Chemical methods for inactivating TSE prion proteins includetreatment of infected material with sodium hydroxide or sodiumhypochlorite solutions (Taylor et al. (1994) Arch. Virol. 139,pp313-326), although infectivity of the prion is shown to surviveexposure to 2M sodium hydroxide for up to two hours.

[0009] Alternative methods for inactivating TSE prions includeautoclaving, but again BSE and scrapie agent has been shown to survivetreatment at 134-138° C. for 18 minutes (Taylor et al. ibid). Thus, acombined chemical/heating approach has been proposed in which infectedmaterials are exposed to 1M sodium hydroxide followed by autoclaving at121° C. for between 30 and 60 minutes (Taylor, J. Hosp. Infect. (1999)43 supplement, pp S69-76). This combined method has shown thatinactivation of TSE infectious agents can be achieved, albeit under veryharsh conditions.

[0010] However, many materials, such as plastics, polymers andnon-protein animal derivatives, cannot be exposed to such extremeconditions without themselves being destroyed. The chemical and physicalprocesses described above are only really suitable for sterilising metalinstruments and surgical tools that are not too large in size and whichcan fit inside a standard autoclave. More delicate instruments such asendoscopes cannot be exposed to extreme conditions of high temperaturewithout the risk of permanent damage to their internal components.

[0011] Further, chemical processes typically involve the use of causticand/or chaotropic agents which are hazardous to handle and dispose of.It would, therefore, be desirable to provide a method for inactivatingagents such as TSE without the need to use large amounts of hazardoussubstances and which method could be scaled up for use on larger objectsand areas as well as on smaller objects.

[0012] Taylor (Vet. J. (2000)159 pp10-17) describes tests usingproteolytic enzymes to deactivate prion proteins. Proteases such astrypsin have little effect in non-denaturing conditions (Taylor (2000)p. 14) but other proteases such as proteinase K may have an effect onTSE infectivity following prolonged digestion times. However, themajority of current TSE inactivation methods are aimed towards chemicaland physical degradation procedures.

[0013] It is an object of the invention, therefore, to provide methodsand means for effectively inactivating TSE infectious agents underconditions that can be readily applied to a variety of locations andsituations. It is a further object of the invention to reduce the needfor extreme conditions of very high temperature and harsh chemicaldenaturants in order to inactivate TSE agents located on or within TSEinfected materials.

[0014] A first aspect of the invention provides a method forinactivating a TSE agent comprising exposing the TSE agent to athermostable proteolytic enzyme.

[0015] The methods and compositions of the invention are suitable forthe inactivation of TSE agent in apparatus and materials infected orsuspected as being infected with TSE agent. Medical apparatus is takento include any item that is in use for surgery, either as an in-patientor out/day patient, dentistry/orthodontics, ophthalmology, gynaecology,obstetrics, veterinary practice, chiropody, audiology, general practice,tattooing. This would include, but not be limited to the following:

[0016] items of small equipment such as scalpels, clamps, forceps,retractors, burrs, probes, needles, picks, scissors, drills, drill bits,chisels, dissectors, rasps, osteotomes, chisels, reamers, curettes,dissectors, shears, suction tubes, scissors, rongeurs, instrument pins,laryngeal mirrors, lead hands, ring cutters, saws, dentist's drills,mirrors, electrodes, irrigation handsets, facoemulsification handsets,

[0017] larger equipment such as endoscopes, laparoscopic instruments,tonometers and other instruments used in invasive procedures,

[0018] furniture such as operating tables, dentist's chairs, lighting,anaesthetic equipment that might be exposed to body tissue duringoperations, and

[0019] equipment such as steam autoclaves, portable autoclaves, porousload sterilisers, sonicators, dishwashers, ultrasonic cleaners, dryingracks.

[0020] Surfaces such as operating theatre walls and floors would also betreated with formulations based on the invention.

[0021] The method of the invention is also suitable for routinedecontamination of instruments and facilities used for slaughter, meathandling, meat rendering, food preparation and associated processes.These would include, but not be limited to, the following:-

[0022] small equipment such as knifes, axes, meat hooks, hand saws,mechanical saws, cleavers,

[0023] larger equipment such as mincers, dicers, rendering equipment,butchers blocks, and

[0024] facilities such as slaughterhouses, butchers, rendering plants.

[0025] In addition, the methods and compositions of the invention aresuitably used in a prophylactic or precautionary mode, where definiteknowledge of infection is uncertain. For example, the method of theinvention can be easily incorporated into the standard sterilisationprotocols used for preparation of surgical apparatus prior to use itsuse in surgical procedures.

[0026] The methods and compositions of the present invention are alsosuitably used for the inactivation of TSE agents in potentiallycontaminated clinical waste and culled animal material. At present, thiswaste material is incinerated at 1000° C., which requires specialisedfacilities and is expensive. It is an advantage of the present inventionthat a TSE inactivation procedure can occur at temperatures and inconditions which do not require highly specialised facilities and thatthe prospects of complete inactivation of the TSE agent are comparableto the more energy intensive and expensive incineration procedures.

[0027] The term transmissible spongiform encephalopathy (TSE) agent isintended to encompass all neurological diseases that are apparentlytransmitted via a pathogenic prion protein intermediate. Such TSEstypically include the human diseases Creutzfeld-Jacob disease (CJD),variant Creutzfeld-Jacob disease (vCJD), Kuru, fatal familial insomniaand Gerstmann-Straussler-Scheinker syndrome. Non-human TSEs includebovine spongiform encephalopathy (BSE), scrapie, feline spongiformencephalopathy, chronic wasting disease, and transmissible minkencephalopathy. Given that vCJD is currently understood to be a humanform of BSE, it is apparent that certain TSE agents are capable ofcrossing the species barrier and that novel TSEs from non-bovine sourcescould become evident in future.

[0028] The proteolytic enzyme of the invention is typically a proteasebut can be suitably any biological polymeric molecule capable ofcatalysing cleavage of a polypeptide chain.

[0029] It is a feature of the invention that the proteolytic enzyme is athermostable enzyme, that is, that it demonstrates optimal biologicalactivity at temperatures in excess of the normal mammalian bodytemperature of 37° C. In embodiments of the invention the enzyme isthermally stable and biologically active, and inactivation is carriedout, at temperatures equal to or greater than 40° C.; preferably in therange of 50° C. to 120° C.; and more preferably where the temperature isbetween 55° C. and 85° C. In a specific embodiment of the invention thetemperature is about 60° C. In a further specific embodiment thetemperature is about 80° C.

[0030] Thermostable proteolytic enzymes suitable for use in the methodsand compositions of the invention are obtainable from a number ofsources such as thermophilic bacteria and archaea. In one embodiment ofthe present invention the thermostable proteolytic enzyme is isolatedfrom thermophilic bacteria, hyperthermophilic bacteria and archaea.Suitable organisms for extraction of proteolytic enzymes for use in theinvention include Thermotoga maritima; Thermotoga neopolitana;Thermotoga thermarum; Fervidobacterium islandicum; Fervidobacteriumnodosum; Fervidobacterium pennivorans; Thermosipho africanus; Aeropyrumpemix; Thermus flavus; pyrococcus spp.; Sulfolobus solfataricus;Desulfurococcus; Bacillus thermoproteolyticus; Bacillusstearo-thermophilus; Bacillus sp. 11231; Bacillus sp. 11276; Bacillussp. 11652, Bacillus sp. 12031; Thermus aquaticus; Thermus caldophilus;Thermus sp. 16132, Thermus sp. 15673; and Thermus sp. Rt41A.

[0031] The aforementioned organisms are not the only sources ofthermostable proteases. Indeed, some organisms that are not consideredto be truly thermophilic can also express thermostable proteolyticenzymes. Such organisms are commonly termed thermodurable in thatalthough they do not choose to live in conditions of high temperature,they can withstand high temperatures for limited periods. A number ofBacillus species fall in the category of thermodurability and are knownto produce thermostable subtilisin-type proteases.

[0032] The pH at which the inactivation is performed can range from acidto alkaline, but is typically in the region of pH 8-13, preferably pHgreater than 9 and more preferably around pH 12. Similarly, thethermostable protease is active in a pH range from acid to alkaline, buttypically is optimally active in the region of pH 8-13, and preferablypH greater than 9 and more preferably around pH 12.

[0033] In an example of the invention in use, the proteolytic enzyme isextracted from a culture of the thermophilic bacteria or archaea. Theculture is suitably maintained under the optimal conditions for theorganism typically within a bioreactor. Thus, a continuous source of theorganism can be maintained, allowing proteolytic enzyme to be obtainedwhenever needed.

[0034] Alternatively, in an example of the invention in use described inmore detail below, the gene encoding a thermostable proteolytic enzymeis isolated from the source organism, Bacillus thermoproteolyticus. Thegene is used to generate a recombinant expression construct, typically aplasmid, which is transformed into a host organism, Escherichia coli.The transformed E. coli is grown in a bioreactor and when at anappropriate cell density the expression of the plasmid construct isinitiated and the proteolytic enzyme harvested using standard methods.The recombinant expression route allows for the production of theproteolytic enzyme product under less extreme conditions of temperaturethan would be required for the original source organism.

[0035] The recombinant route is further advantageous in that it allowsfor the genetic manipulation of recombinant thermostable protease genesin order to increase thermal stability or biological activity or forsome other purpose. Thus, the activity of a thermostable proteolyticenzyme can be readily optimised for use in the methods and compositionsof the invention.

[0036] Proteases are proteolytic enzymes that are carbonyl hydrolaseswhich generally act to cleave peptide bonds of proteins or peptides. Asused herein, “protease” means a naturally-occurring protease or arecombinant protease. Naturally-occurring proteases includea-aminoacylpeptide hydrolase, peptidylamino acid hydrolase, acylaminohydrolase, serine carboxypeptidase, metallocarboxypeptidase, thiolproteinase, carboxylproteinase and metalloproteinase. Serine, metallo,thiol and acid proteases are included, as well as endo andexo-proteases.

[0037] The present invention includes the use of protease enzymes, forexample naturally occurring carbonyl hydrolases or non-naturallyoccurring carbonyl hydrolase variants (protease variants). The proteaseenzymes useful in this invention exhibit a greater hydrolytic activitythan proteinase K. In the context of this invention, the hydrolyticactivity of the subtilisins was measured and comparable assays include,but are not limited to those described in Proteolytic enzymes, PracticalApproach (Ed. by Beynon, R J and Bond, J S, Oxford University Press, NewYork, Oxford, pp. 25-55 (1989); and the digestion of PrP-res proteins(Raymond, et al, Nature, 388:285-288 (1997). For example, the enzymaticactivity of subtilisins could also be measured by using chromogenicsubstrates. Incubation of proteases with these substrates could resultin the cleavage of the substrate and liberation of p-nitroaniline thatis detected spectrophotometrically at 405 nm. Other exemplary methods ofanalyzing the subtilisins are by using the substrateN-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (0.8 mM in 20 mM sodiumphosphate buffer, pH 8.5 or 0.8 mM in 20 mM Britton-Robinson buffer, pH8.5). The incubation is carried out at 25° C. and followedspectrophotometrically form 4 min. The concentration of the protease ischosen so that the liberation of p-nitroaniline is linear during thewhole analysis.

[0038] Proteases useful in the practice of this invention include allthose disclosed in U.S. Pat. No. 6,312,936, the contents of which arehereby incorporated by reference, e.g. those proteases found in Bacillusamyloliquefaciens (SEQ. ID NO:7), Bacillus subtilis (SEQ. ID. NO:8),Bacillus licheniformis (SEQ.ID.NO:9) and Bacillus lentus (SEQ.ID.NO:10).Another Bacillus lentus useful in the practice of this invention is theDSM 5483. The hydrolase variants may also have a different proteolyticactivity, stability, substrate specificity, pH profile and/orperformance characteristic as compared to the precursor carbonylhydrolase from which the amino acid sequence of the variant is derived.Specifically, such protease variants have an amino acid sequence notfound in nature, which is derived by substitution of a plurality ofamino acid residues of a precursor protease with different amino acids.The precursor protease may be a naturally-occurring protease or arecombinant protease.

[0039] The protease variants useful herein encompass the substitution ofany of the nineteen naturally occurring L-amino acids at the designatedamino acid residue positions. Such substitutions can be made in anyprecursor subtilisin (procaryotic, eucaryotic, mammalian, etc.).Throughout this application reference is made to various amino acids byway of common one- and three-letter codes. Such codes are identified inDale, M. W. (1989), Molecular Genetics of Bacteria, John Wiley & Sons,Ltd., Appendix B.

[0040] The protease variants useful herein are preferably derived from aBacillus subtilisin. More preferably, the protease variants are derivedfrom Bacillus amyloliquefaciens subtilisin, Bacillus lentus subtilisinand/or subtilisin 309.

[0041] Subtilisins are bacterial or fungal proteases which generally actto cleave peptide bonds of proteins or peptides. As used herein,“subtilisin” means a naturally-occurring subtilisin or a recombinantsubtilisin. A series of naturally-occurring subtilisins is known to beproduced and often secreted by various microbial species. Amino acidsequences of the members of this series are not entirely homologous.However, the subtilisins in this series exhibit the same or similar typeof proteolytic activity. This class of serine proteases shares a commonamino acid sequence defining a catalytic triad which distinguishes themfrom the chymotrypsin related class of serine proteases. The subtilisinsand chymotrypsin related serine proteases both have a catalytic triadcomprising aspartate, histidine and serine. In the subtilisin relatedproteases the relative order of these amino acids, reading from theamino to carboxy terminus, is aspartate-histidine-serine. In thechymotrypsin related proteases, the relative order, however, ishistidine-aspartate-serine. Thus, subtilisin herein refers to a serineprotease having the catalytic triad of subtilisin related proteases.Examples include but are not limited to the subtilisins identified inFIG. 15 herein. Generally and for purposes of the present invention,numbering of the amino acids in proteases corresponds to the numbersassigned to the mature Bacillus amyloliquefaciens subtilisin sequencepresented in FIG. 13.

[0042] “Recombinant subtilisin” or “recombinant protease” refer to asubtilisin or protease in which the DNA sequence encoding the subtilisinor protease is modified to produce a variant (or mutant) DNA sequencewhich encodes the substitution, deletion or insertion of one or moreamino acids in the naturally-occurring amino acid sequence. Suitablemethods to produce such modification, and which may be combined withthose disclosed herein, include those disclosed in U.S. Pat. No. RE34,606, U.S. Pat. No. 5,204,015 and U.S. Pat. No. 5,185,258, U.S. Pat.No. 5,700,676, U.S. Pat. No. 5,801,038, and U.S. Pat. No. 5,763,257.

[0043] “Non-human subtilisins” and the DNA encoding them may be obtainedfrom many procaryotic and eucaryotic organisms. Suitable examples ofprocaryotic organisms include gram negative organisms such as E. coli orPseudomonas and gram positive bacteria such as Micrococcus or Bacillus.Examples of eucaryotic organisms from which subtilisin and their genesmay be obtained include yeast such as Saccharomyces cerevisiae, fungisuch as Aspergillus sp.

[0044] A “protease variant” has an amino acid sequence which is derivedfrom the amino acid sequence of a “precursor protease”. The precursorproteases include naturally-occurring proteases and recombinantproteases. The amino acid sequence of the protease variant is “derived”from the precursor protease amino acid sequence by the substitution,deletion or insertion of one or more amino acids of the precursor aminoacid sequence. Such modification is of the “precursor DNA sequence”which encodes the amino acid sequence of the precursor protease ratherthan manipulation of the precursor protease enzyme perse. Suitablemethods for such manipulation of the precursor DNA sequence includemethods disclosed herein, as well as methods known to those skilled inthe art (see, for example, EP 0 328299, WO89/06279 and the U.S. patentsand applications already referenced herein).

[0045] In a preferred embodiment of the invention, the protease is asubtilisin derived from a Bacillus species, to include, but not limitedto B. subtilis, B. lentus, B. licheniformis and B. amyloliquefaciens.

[0046] In a further embodiment, the protease is a Bacillus lentussubtilisin having mutations N76D, S103A and V104I described previouslyin WO 95/10615 and identified specifically in that patent as SEQ. ID.NO. 12 and shown in FIG. 7.

[0047] In a preferred embodiment, the protease used for inactivation ofTSE agents on surgical instruments or in waste animal material is amodified Bacillus subtilis subtilisin having equivalent amino acidchanges to those described for the Bacillus lentus subtilisin variant;specifically amino acid changes N76D, S103A and V104I. This protease isreferred to as MC-3 in Examples 3 and 4 below.

[0048] In a further embodiment, the subtilisin used for the inactivationof TSE was the subtilisin from Bacillus licheniformis referred to asMC-4 in Examples 3 and 4 below. Equivalent mutations to those describedfor Bacillus subtilis and Bacillus lentus subtilisins would also bevaluable reagents for inactivation of TSEs. Similarly, the subtilisinfrom B. amyloliquefaciens (often referred to as BPN′) carrying mutationsN76D, S103A and V104I is also a suitable protease for theseapplications.

[0049] One embodiment of the present invention utilizes proteasevariants having at least one modification of an amino acid positioncorresponding to positions 27, 76, 87, 101, 103, 104, 123, 159, 222,232, 236, 245, 248, 252, and 274 of Bacillus amyloliquefacienssubtilisin. Exemplary embodiments and/or combinations contemplated bythe inventors include Y217L; K27RN104Y/N123S/T274A; N76D/S103A/V104I ;S101G/S103A/V104I/G159D/A232V/Q236H/Q245R/N248D/N252K. Other embodimentsinclude at least one modification of the precursor protease made to atleast one position corresponding to positions 120, 167, 170, 194, 195,and 235 of Bacillus amyloliquefaciens Exemplary embodiments includecombinations selected from G195E/M222A; M222S; Y167A/R170S/A194P;D36_N76D/H120D/G195E/K235N. Still another embodiment includes at leastone modification at an amino acid position corresponding to positionsselected from the group consisting of 3, 4, 99, 101, 103, 104, 159, 194,199, 205, 217 of Bacillus lentus. Exemplary embodiments includecombinations selected from S99D/S101R/S 103A/V104I/G159S;S99D/S101R/S103A/V104I/G159S/S3T/V4I/A194P/V199M/V205I/L217D andS99D/S101R/S103A/V104I/G159S/S3T/V4I/V205I/L217D of a mature Bacilluslentus DSM 5483 alkaline protease. These amino acid position numbersrefer to those assigned to the mature Bacillus amyloliquefacienssubtilisin sequence presented in FIG. 13. The invention, however, is notlimited to the mutation of this particular subtilisin but extends toprecursor proteases containing amino acid residues at positions whichare “equivalent” to the particular identified residues in Bacillusamyloliquefaciens subtilisin. In a preferred embodiment of the presentinvention, the precursor protease is selected from a Bacillusamyloliquefaciens or a Bacillus lentus subtilisin, the substitutions aremade at the equivalent amino acid residue positions in B. lentuscorresponding to those listed above.

[0050] A residue (amino acid) position of a precursor protease isequivalent to a residue of Bacillus amyloliquefaciens subtilisin if itis either homologous (i.e., corresponding in position in either primaryor tertiary structure) or analogous to a specific residue or portion ofthat residue in Bacillus amyloliquefaciens subtilisin (i.e., having thesame or similar functional capacity to combine, react, or interactchemically).

[0051] In order to establish homology to primary structure, the aminoacid sequence of a precursor protease is directly compared to theBacillus amyloliquefaciens subtilisin primary sequence and particularlyto a set of residues known to be invariant in subtilisins for whichsequence is known. For example, FIG. 14 herein shows the conservedresidues as between B. amyloliquefaciens subtilisin and B. lentussubtilisin. After aligning the conserved residues, allowing fornecessary insertions and deletions in order to maintain alignment (i.e.,avoiding the elimination of conserved residues through arbitrarydeletion and insertion), the residues equivalent to particular aminoacids in the primary sequence of Bacillus amyloliquefaciens subtilisinare defined. Alignment of conserved residues preferably should conserve100% of such residues. However, alignment of greater than 75% or aslittle as 50% of conserved residues is also adequate to defineequivalent residues. Conservation of the catalytic triad,Asp32/His64/Ser221 should be maintained. Siezen et al. (1991) ProteinEng. 4(7):719-737 shows the alignment of a large number of serineproteases. Siezen et al. refer to the grouping as subtilases orsubtilisin-like serine proteases.

[0052] For example, in FIG. 15, the amino acid sequence of subtilisinfrom Bacillus amyloliquefaciens, Bacillus subtilis, Bacilluslicheniformis (carlsbergensis) and Bacillus lentus are aligned toprovide the maximum amount of homology between amino acid sequences. Acomparison of these sequences shows that there are a number of conservedresidues contained in each sequence. These conserved residues (asbetween BPN′ and B. lentus) are identified in FIG. 14.

[0053] These conserved residues, thus, may be used to define thecorresponding equivalent amino acid residues of Bacillusamyloliquefaciens subtilisin in other subtilisins such as subtilisinfrom Bacillus lentus (PCT Publication No. WO89/06279 published Jul. 13,1989), the preferred protease precursor enzyme herein, or the subtilisinreferred to as PB92 (EP 0 328 299), which is highly homologous to thepreferred Bacillus lentus subtilisin. The amino acid sequences ofcertain of these subtilisins are aligned in FIGS. 15A and 15B with thesequence of Bacillus amyloliquefaciens subtilisin to produce the maximumhomology of conserved residues. As can be seen, there are a number ofdeletions in the sequence of Bacillus lentus as compared to Bacillusamyloliquefaciens subtilisin. Thus, for example, the equivalent aminoacid for Val165 in Bacillus amyloliquefaciens subtilisin in the othersubtilisins is isoleucine for B. lentus and B. licheniformis.

[0054] “Equivalent residues” may also be defined by determining homologyat the level of tertiary structure for a precursor protease whosetertiary structure has been determined by x-ray crystallography.Equivalent residues are defined as those for which the atomiccoordinates of two or more of the main chain atoms of a particular aminoacid residue of the precursor protease and Bacillus amyloliquefacienssubtilisin (N on N, CA on CA, C on C and O on O) are within 0.13 nm andpreferably 0.1 nm after alignment. Alignment is achieved after the bestmodel has been oriented and positioned to give the maximum overlap ofatomic coordinates of non-hydrogen protein atoms of the protease inquestion to the Bacillus amyloliquefaciens subtilisin. The best model isthe crystallographic model giving the lowest R factor for experimentaldiffraction data at the highest resolution available.${R\quad {factor}} = \frac{{\sum_{h}{{\_ Fo}(h)\_}} - {{\_ Fc}(h)\_}}{\sum_{h}{{\_ Fo}(h)\_}}$

[0055] Equivalent residues which are functionally analogous to aspecific residue of Bacillus amyloliquefaciens subtilisin are defined asthose amino acids of the precursor protease which may adopt aconformation such that they either alter, modify or contribute toprotein structure, substrate binding or catalysis in a manner definedand attributed to a specific residue of the Bacillus amyloliquefacienssubtilisin. Further, they are those residues of the precursor protease(for which a tertiary structure has been obtained by x-raycrystallography) which occupy an analogous position to the extent that,although the main chain atoms of the given residue may not satisfy thecriteria of equivalence on the basis of occupying a homologous position,the atomic coordinates of at least two of the side chain atoms of theresidue lie with 0.1 3 nm of the corresponding side chain atoms ofBacillus amyloliquefaciens subtilisin. The coordinates of the threedimensional structure of Bacillus amyloliquefaciens subtilisin are setforth in EPO Publication No. 0 251 446 (equivalent to U.S. Pat. No.5,182,204, the disclosure of which is incorporated herein by reference)and can be used as outlined above to determine equivalent residues onthe level of tertiary structure.

[0056] Some of the residues identified for substitution are conservedresidues whereas others are not. In the case of residues which are notconserved, the substitution of one or more amino acids is limited tosubstitutions which produce a variant which has an amino acid sequencethat does not correspond to one found in nature. In the case ofconserved residues, such substitutions should not result in anaturally-occurring sequence. The protease variants of the presentinvention include the mature forms of protease variants, as well as thepro- and prepro-forms of such protease variants. The prepro-forms arethe preferred construction since this facilitates the expression,secretion and maturation of the protease variants.

[0057] A second aspect of the invention provides for a method ofsterilising apparatus, comprising the step of exposing the apparatus toa solution comprising a thermostable proteolytic enzyme.

[0058] The term “sterilising” is commonly understood to mean theprocedure by which living organisms are removed from or killed in asubstrate, such as a piece of equipment or a solution. In the presentcase the TSE agent, or prion, is not technically considered to be aliving organism, in the sense that a bacterium or virus is, because itdoes not apparently contain any genetic material. However, thetransmission of the TSE pathogenic agent between animals does result indisease. Thus, the term “sterilising” as used herein is applied to theprocedure by which both pathogenic agents (such as TSE agents) andliving organisms are rendered non-infective or removed from or killed ina substrate.

[0059] In a preferred embodiment, the method of the invention comprisesmaintaining the sterilising solution at a temperature below 100° C.,preferably at least 45° C. and more preferably between 45° C. and 85° C.The pH of the sterilising solution can range from acid to alkaline, butis typically in the region of pH 8-13, at least pH 9 and more preferablyaround pH 12. Similarly, the thermostable protease is active in a pHrange from acid to alkaline, but typically is optimally active in theregion of pH 8-13, at least pH9 and more preferably around pH 12.

[0060] In specific embodiments of the invention the sterilising solutionis applied to the apparatus as a spray. The advantage of this mode ofapplication is that, larger surface areas of apparatus, operating tablesor even walls of rooms (for example in abattoirs) can be treated withthe sterilising solution of the invention. Typically the solution willbe heated to an optimal temperature, for example between 60° C. to 80°C., before being sprayed onto the surface that is to be sterilised, thatsurface being optionally heated in advance.

[0061] Alternatively, the apparatus is immersed in the sterilisingsolution for a predetermined period of time. Again, the temperature ofthe solution is typically optimised prior to immersion of thecontaminated apparatus. It is optional to include ultrasonication meansin the immersion bath to enable ultrasonic cleaning to occur at the sametime as treatment with the sterilising solution of the invention.

[0062] In a third aspect, the invention provides for a method ofsterilising material, comprising exposing said material to a firstsolution comprising a thermostable proteolytic enzyme; and then exposingthe apparatus to at least a second solution comprising a secondthermostable proteolytic enzyme. In use, the material is typicallyapparatus, surgical or meat rendering equipment, or TSE infectedbiological waste.

[0063] By dividing the sterilisation method into at least two, andoptionally more, successive steps the conditions in each step can beoptimised to ensure maximum inactivation of any TSE agent present. Thus,the temperatures and/or pH of successive steps can be different. Inspecific embodiments of the invention the proteolytic enzymes in thefirst and second (and optionally more) solutions are the same, or aredifferent.

[0064] In a fourth aspect the invention provides for a composition forinactivating a TSE agent, comprising a thermostable proteolytic enzyme.

[0065] Typically, the composition of the invention further comprises abuffering agent. In a specific embodiment of the invention the bufferingagent has a pK_(a) of between 8 and 13. Alkaline buffers suitable foruse in the method of the invention include4-cyclohexylamino-1-butanesulfonic acid (CABS) which has a pKa of 10.7at 25° C., and 3-cyclohexylamino-1-propanesulfonic acid (CAPS) which hasa pKa of 10.4 at 25° C.

[0066] Alternatively, the composition of the invention comprisessufficient sodium hydroxide or other alkaline agent to adjust the pH ofthe composition to alkaline, preferably to at least pH 9 and morepreferably around pH 12. Addition of 1M sodium hydroxide to thecomposition of the invention, using a pH probe calibrated usingUniversal standards, is generally sufficient to set the pH of thecomposition to around 12.

[0067] Further provided by the invention is apparatus for inactivating aTSE agent comprising:

[0068] a. a chamber for receiving contaminated material;

[0069] b. means for controlling the temperature of the chamber; and

[0070] c. a thermostable proteolytic enzyme active at alkaline pH,located within the chamber, the chamber optionally containing a solutionof the thermostable proteolytic enzyme at a temperature of 45° C. to 85°C.

[0071] Further aspects of the invention provide for uses of theaforementioned compositions for the inactivation of TSE agents.

[0072] An advantage of the invention is its use in degrading TSE andsimilar agents, and it has been found in operation of particularembodiments of the invention that TSE-contaminated material has beensuccessfully decontaminated using a combination of elevated temperatureof around 50° C. to 70° C. and alkaline pH of around 9 to 12, with athermostable, alkophilic proteolytic enzyme. Whilst it is on occasionpossible to achieve some decontamination by extremely high temperaturealone, it is of significant benefit to be able to inactivate TSE whilstavoiding extreme conditions, such as extremes of temperature, which leadto damage to the equipment being decontaminated

[0073] In a separate, though related, aspect of the present invention,the problem of detecting infective material is addressed. If it werepossible to test an item of equipment for contamination either prior toor after carrying out a sterilisation process, then this would be ofsignificant utility.

[0074] An anti-prion antibody, mAb 6H4 is available commercially fromPrionics, Switzerland. This antibody can be used to detect prionprotein, detected typically using a second antibody conjugated to adetectable marker, which second antibody binds to the first.

[0075] A difficulty that has been discovered by the present inventors isthat binding of this antibody to equipment suspected of beingcontaminated with prion, or equipment that is suspected to becontaminated but which has been subjected to treatment intended todestroy the prion, does not correlate with infectivity. It has, forinstance, been discovered by the inventors that prion-infected mousebrain homogenate, digested with protease, and run on SDS-PAGE, thenprobed with anti-prion antibody, shows a negative result, that is to sayabsence of antibody binding. This material nevertheless retainsinfectivity.

[0076] A further object of the invention is to provide methods andreagents for identifying infective prion material and for determiningwhen infective material has been removed by treatment.

[0077] Accordingly, a further aspect of the invention provides a methodof examining prion-infected or suspected prion-infected material anddetermining whether dimers of prion protein are present.

[0078] This aspect of the invention is based upon a correlation betweenpresence of dimers of prion protein and presence of infectivity. As theprion protein can exist in a number of different glycosylation states,references to dimer is intended to include dimers of the protein whetherone or other or both is in any of a number of different possibleglycosylation states. Reference to dimer is also intended to includereference to dimers of fragments of prion protein, thosefragment-containing dimers retaining infectivity. Proteolytic treatmentof the prion can result in removal of part of the protein sequence,leaving behind a residue which in dimer form retains infectivity, andthese fragment dimers as well as prion-prion fragment dimers andfragment heterodimers are included within the definition of dimer in thepresent aspect of the invention.

[0079] The method enables detection of presence of dimer and henceenables detection of presence of infective material in cases whereprevious tests, designed to identify whether the monomer is present,would have given a negative result whereas in fact infectivity remainedin that particular sample being tested. Hence, one advantage of themethod is that at least some of the previous false negative results areeliminated. Avoiding these false negatives is clearly of significantvalue in testing alleged contaminated material as well as in testingmaterial after treatment intended to remove infectivity.

[0080] As set out in more detail in an example below, prion dimers canbe detected by using an antibody that binds to a prion monomer, using asecond antibody, which has been labelled, to bind to the anti-prionantibody, and determining whether a protein is identified havingapproximately twice the molecular weight of the expected molecularweight of the prion monomer. As mentioned, the prion can be glycosylatedin a number of different positions, typically 0, 1 or 2 positions, sowhen Western blotting is used, a number of bands may be seen on theresultant blot, corresponding to protein molecules having differentglycosylation patterns. A known monomer is expected to have a molecularweight of about 33-35 kDa at its full length, however, it is routinelyobserved on SDS-PAGE gels and immunoblots as a limited proteolyticcleavage product known as “PrP 27-30”. Correspondingly, the dimer isexpected to be found at that part of the blot corresponding to about54-70 kDa.

[0081] The dimer forms may be probed using an antibody specific to thedimer, that is to say an antibody which binds to the dimer butsubstantially does not bind to the monomer form of the prion. Thisantibody may itself be labelled or may be probed using a secondarylabelled antibody.

[0082] A preferred method of this aspect of the invention thus comprisesa method of detecting prion infectivity comprising detecting prion dimerin a sample.

[0083] The invention additionally provides reagent for detecting priondimers, and thus a further embodiment of the invention lies in anantibody specific for prion dimer, which antibody binds prion dimer butsubstantially does not bind prion monomer. This antibody optionally islabelled, and in an example described below the label is horseradishperoxidase; other suitable labels are alkaline phosphatase, betagalactosidase and D-biotin, though any suitable label may be used.

[0084] An alternative reagent for detection of presence of dimercomprises an antibody that binds to both monomer and a dimer and amolecular weight standard, such as one that corresponds to the molecularweight of the dimer. These are suitably used in combination with, forexample, Western blotting techniques, to identify a protein having amolecular weight approximately twice that of the prion monomer. Allthese reagents are suitably incorporated into prion dimer detectionkits.

[0085] To make an antibody selective for prion dimer, an animal isimmunised with prion dimer and then serum extracted from the animal isrun on a prion dimer column to identify antibodies that bind the dimer.These are then tested for cross-reactivity with prion monomer, withcross-reacting antibodies removed. The removal can be effected using acolumn loaded with prion monomer, the antibodies that emerge therefromthen being tested for absence of cross-reactivity.

[0086] Isolated prion dimer forms a further embodiment of the invention.This isolated material can be obtained simply by cutting out a portionof the SDS-PAGE gel used to resolve prion dimers. Alternatively, otherseparation techniques can be used to extract the prion dimer fromhomogenised prion-infected mouse brain. Antibodies that bind to theprion monomer and which are cross reactive can be used to confirm thatthe material thus obtained is prion dimer and not other protein of thesame molecular weight.

[0087] As set out in more detail in the examples, prion strain 301V, amouse passaged isolate, derived from a Holstein-Fresian cow terminallyill with BSE is used as an example of a prion strain. Infection is knownto be produced by intracerebral inoculation and the incubation periodrequired for the onset of clinical symptoms is remarkably uniform. Thatis to say, providing that the dose of infectious agent is sufficientthen the classic signs of disease will appear at a defined timepost-inoculation (in VM mice this is 120 days). For obvious reasonsno-one has tested these properties on humans, however, the mousebioassay is regarded as the closest available model and therefore a goodindicator of BSE infection in man.

[0088] Using the Western blot detection system, in an example below, themonomer is apparently completely removed by protease digestion. Underthese conditions, however, samples which were subsequently used in vivodid not prevent infection and may even have enhanced its onset. Theremust, therefore, be another source of infection other than the monomer.Since the monomer can be removed (or at worst dramatically reduced inconcentration) this indicates a primary role in infectivity for thedimer—either alone or in combination with the monomer.

[0089] The second aspect of the invention thus also provides dimerremoval using protease degradation according to the first aspect whichis optionally enhanced by environmental conditions—high temperature,extremes of pH, and/or the use of detergents (e.g. SDS). As well asusing single enzyme treatments, combinations of proteases and/or otherenzymes can be used. For example, better degradation of the infectiveagent may be achieved by the addition of lipases, peptidases,glycosylases, nucleases and other enzymes.

[0090] To confirm dimer removal, a dimer cross-reactive antibody can beused in conjunction with a suitable detection system, one example beinga sensitive in vitro detection system currently available fromInvitrogen, referred to as Western Breeze, to confirm removal prior tofurther mouse bioassays.

[0091] The methods and compositions of specific embodiments of theinventions are described in more detail below and are illustrated by theaccompanying drawings and tables in which;

[0092]FIG. 1 shows the effect of temperature on a protease M digest ofmouse brain homogenate (mbh);

[0093]FIG. 2 shows the effect of pH on a protease M digest of mbh;

[0094]FIG. 3 shows a Bacillus thermoproteolyticus Rokko digest of mbh;

[0095]FIG. 4 shows the effect of sodium dodecyl sulfate (SDS) on Rokkodigest of mbh;

[0096]FIG. 5 shows and SDS-PAGE of mbh;

[0097]FIG. 6 shows and immunoblot of mbh;

[0098]FIG. 7 shows a protease G, R and C digest of mbh;

[0099]FIG. 8 shows an immunoblot of the digest in FIG. 7;

[0100] FIGS. 9 to 12 show blots of BSE (301V)-infected mouse brainhomogenate, to illustrate correlation of infectivity with prion dimer,and as further explained in the examples below;

[0101] FIGS. 13A-C depict the DNA (SEQ.ID.NO:1) and amino acid sequence(SEQ.ID.NO:2) for Bacillus amyloliquefaciens subtilisin and a partialrestriction map of this gene.

[0102]FIG. 14 depicts the conserved amino acid residues amongsubtilisins from Bacillus amyloliquefaciens (BPN)′ and Bacillus lentus(wild-type).

[0103]FIGS. 15A and 15B depict the amino acid sequence of foursubtilisins. The top line represents the amino acid sequence ofsubtilisin from Bacillus amyloliquefaciens subtilisin (also sometimesreferred to as subtilisin BPN′) (SEQ.ID.NO: 7). The second line depictsthe amino acid sequence of subtilisin from Bacillus subtilis (SEQ.ID.NO:8). The third line depicts the amino acid sequence of subtilisin from B.licheniformis (SEQ.ID.NO: 9). The fourth line depicts the amino acidsequence of subtilisin from Bacillus lentus (SEQ.ID.NO:10). The symbol *denotes the absence of specific amino acid residues as compared tosubtilisin BPN′.

[0104]FIG. 16 shows an MC-A, MC-3 and MC-4 digest of mbh.

[0105]FIG. 17 shows a comparison of MC-A, MC-3 and MC-4 mbh digests witha Properase mbh digest.

[0106]FIG. 18 also shows a comparison of MCA, MC-3 and MC4 mbh digestswith a Properase mbh digest.

[0107]FIG. 19 shows a temperature profile of MC-3.

[0108]FIG. 20 shows detection of MC-A, MC-3 and MC4 mbh digests withPAb2.

[0109]FIG. 21 shows MC-3 dilutions at pH 10 and pH 12.

[0110]FIG. 22 shows a comparison of MC-A, MC-3 and MC-4 mbh digests witha Proteinase K mbh digest,

[0111] Table 1 shows the incubation period of VM mice infected with BSE(301V)-infected mbh,

[0112] Table 2 shows the incubation period of VM mice infected with BSE(301V)-infected mbh spiked into a background of meat and bonemeal (mbm).

[0113] Table 3 shows organisms from which thermostable proteases wereanalysed.

EXAMPLES Example 1

[0114] VM Mouse Colony and Incubation with BSE (301V) Agent

[0115] Studies on the inactivation of the TSE agent, BSE strain (301V),required the establishment of a mouse breeding colony for the generationof both uninfectious and infectious brain homogenate (mbh) and itssubsequent titration and bioassay. The VM mouse strain selected for usein the study was obtained from Dr David Taylor (Institute of AnimalHealth, Edinburgh). Six pairs were introduced into a dedicated roomwithin an animal facility. Mice were screened for their health statusand a breeding programme initiated.

[0116] BSE (301V) infectious mouse brain (IAH, Edinburgh) was preparedfor inoculation by crude homogenisation followed by passage of thebrains through increasingly fine gauge luer-locked needles (from21G-27G) to and from a contained safety syringe into a closedseptum-topped vial. This procedure was carried out in a validated safetycabinet within a containment level 3 (CL3) laboratory immediately priorto intracerebral inoculation of the VM mice. The anaesthetised(alphadolone/alphaxalone) mice were inoculated intracerebrally via 26gauge needles with 20 microlitres of the mouse brain homogenatepreparation. Forty-nine out of fifty mice survived this procedure. Thesewere retained to allow incubation of the agent and the generation of therequired quantity of high-titre infectious material.

[0117] Biomass Production

[0118] A wide variety of organisms were chosen for the production ofbiomass in order to provide as broad a selection of thermostableproteolytic enzymes as possible. Organisms selected ranged from thosegrowing optimally at moderately thermophilic temperatures (50° C.)through to extremely thermophilic temperatures (100° C.) and includedmembers of both the Archaea and the Bacteria. Thermophiles were alsoselected to cover a wide range of growth pH, encompassing pH optima frompH2.5 to pH11.5. The majority of organisms were grown in batch culture,however, where the growth requirements of the organism were particularlyfastidious, continuous culture was used (Raven and Sharp (1997) AppliedMicrobial Physiology: A Practical Approach, Ch. 2, Eds. Stanbury andRhodes, OUP pp.23-52). Depending on the biomass yield of the organismbeing grown, batch culture volumes of between 20L and 120L were employedto achieve the desired amount of cell paste. The continuous culturesystem utilised either a 2L or 5L working volume gas lift bioreactorconstructed entirely of glass and PTFE. More prolific organisms such asBacillus spp., and Thermus spp., were pre-screened to select those withhigh levels of protease activity prior to their culture on a largerscale. A quick and sensitive fluorometric protease assay utilisingmicrotitre plates was adopted for this purpose to permit highthrough-put screening (EnzChek™, Molecular Probes, Leiden, Netherlands).

[0119] Culture biomass was harvested by continuous centrifugation(Contifuge Stratos™, Kendro Laboratory Products, Bishop Stortford, UK)and stored at −80° C. Culture supernatants were concentrated with a 10KDa cut off tangential flow filter (Pall filtration, Portsmouth, UK).Proteins were precipitated with ammonium sulphate (90% saturation) andstored at −80° C.

[0120] Protein Purification

[0121] A rapid protease screening and purification technique wasrequired in order to process all of the crude protein preparations afterthe biomass production stage. A dye-ligand affinity chromatographysystem was used for this purpose (PIKSI M™, Affinity ChromatographyLtd., Isle of Man, UK). Initially, each crude ammonium sulphateprecipitate was dissolved in buffer and passed through a desaltingcolumn. Each sample was then loaded onto the PIKSI M test kit, whichcontained 10 different affinity ligands. Fractions were then assayed forprotease activity to determine the most suitable matrix for purificationof the protease, either by positive binding of the target molecule andthen elution, or by negative binding of contaminants. The purificationwas then scaled up using the same affinity matrix in conjunction with anFPLC system (Amersham-Pharmacia Biotech, Amersham, UK). By combiningthis technique with the fluorometric protease assay, the rapid screeningof many fractions could be undertaken.

[0122] The fluorometric protease assay utilises casein derivatives thatare heavily labelled with green fluorescent BODIPY FL in which theconjugates' fluorescence is almost totally quenched. Protease catalysedhydrolysis releases the highly fluorescent label and the resultantfluorescence can be measured on a fluorometric microplate reader(Labsystems Fluoroscan II™). The increase in fluorescence isproportional to protease activity and was compared with that of astandard protease (Protease X, Sigma-Aldrich, Poole, UK).

[0123] Protease Characterisation

[0124] A range of thermostable proteases were analysed (see Table 3).Direct characterisation of activity was carried out using the closestnon-infectious analogue to BSE (301V)—infectious mouse brain homogenateavailable as substrate, i.e. normal VM mouse brain homogenate. Initialdigests of total uninfected mouse brain homogenate (mbh) were performedover thirty minutes at 60° C. and at pH7.0. The samples were then boiledunder reducing conditions and analysed by SDS-PAGE on pre-cast NuPage4-12% Bis-Tris gels (Novex™, San Diego, US). Gels were fixed usingstandard procedures and the proteins visualised using the Novexcolloidal blue staining kit. The results showed both the levels ofactivity of the proteases against the mbh substrate under theseconditions and also the level of purity of individual proteases.Protease concentrations used in the assays were based on total proteinconcentration, therefore, a wide range of activities was observed fromlimited to complete digestion.

[0125] Activity profiles showing the full effect of pH and temperatureon mbh digestion were then produced for each enzyme. Several enzymesshowed extensive activity over the complete range of conditions (pH 2-12and 50-100° C.). In general, however, complete digestion of mbh was onlyachieved over a narrower range indicative of the enzymes' pH andtemperature optima. FIGS. 1 and 2 show the activity profiles for asample protease, Bacillus protease M. As can be seen the enzyme ismoderately thermophilic and gives complete digestion at temperatures upto 70° C. Similarly the pH profile indicates a preference for neutral oralkaline conditions.

[0126] Protease Testing

[0127] Several enzymes did not give complete digestion of mbh even whenincubation times were increased to 24 hours. This may have simply beendue to low protease activity, however, several highly purified andconcentrated enzymes still only produced partial digests. In thesereactions little enzyme-substrate interaction appeared to be occurring.It was considered that this could be due to non-proteinaceous material,e.g. lipids, surrounding the substrate and preventing interaction,however, pre-treatment with lipases and other enzymes had no observableeffect (data not shown). A second possibility considered was the effectof repulsive surface charges between proteases and the substrate. Oneenzyme in particular, purified from Bacillus thermoproteolyticus Rokko,consistently produced poor digestion of mbh even at high concentrations.This enzyme was known to remain active in the presence of high levels ofdetergents, therefore, digests were performed with the addition of SDSin an attempt to overcome this effect. FIGS. 3 and 4 show B.thermoproteolyticus Rokko digests of mbh in the absence and presence ofSDS. FIG. 3 clearly indicates that under standard conditions there islittle difference in digestion even as the protease concentration isincreased to 20 mg.ml⁻¹. On addition of SDS (lanes 2-5), no proteinbands, except the protease itself, are visible indicating a completedigest. Tested proteases could, therefore, be divided simply into threecategories: (i) those able to give total mbh digestion in the absence ofdetergent, (ii) those able to give total mbh digestion in the presenceof SDS and (iii) those unable to digest the substrate fully. Proteasesin categories (i) and (ii) were selected for immediate further study,while those in category (iii) were either rejected or assumed to berequired in greater quantity and/or higher purity.

[0128] Western Blotting of Mouse Brain Homogenate

[0129] Mbh proteins were transferred onto nitrocellulose and blockedovernight in PBST-Tween (PBST)+3% skimmed milk powder. The membrane waswashed (×3 in PBST) and incubated for 1 hour with 6H4 anti-humanrecombinant PrP monoclonal antibody (Prionics, Zurich, Switzerland).After a second washing step, anti-mouse HRP-conjugate was added and themembrane incubated for 1 hour. Washing was repeated and the antibodyreaction visualised by addition of TMB (Harlow and Lane (1988),Antibodies: A Laboratory Manual, Cold Spring Harbor Press).

[0130]FIGS. 5 and 6 show an SDS-PAGE and an immunoblot of undigested mbhrespectively. Although there is some non-specific background, dark bandsindicating the presence of mouse prion can clearly be seen at theexpected molecular weight (˜33-35kDa). Distinct bands are also visibleat ˜66-70kDa, which may correspond to prion dimers previously reported(Safar et al. (1990) Proc. Natl. Acad. Sci. USA, 87:pp6373-6377).

[0131] Western transfer and immunoblotting were used to confirm that noimmunoreactive fragment of mouse PrP^(c) remained after digestion. FIG.7 shows mbh digested to completion with three closely relatedthermostable proteolytic enzymes (Proteases G, R and C), as assessed bySDS-PAGE. In the corresponding immunoblot (FIG. 8) only 2 bands arevisible, both with an apparent MW of ˜23kDa. The strong band in lane 9corresponds to recombinant mouse PrP (+ve control), whilst the weak bandin lane 4 appears to be due to a slight reaction with the heavily loadedenzyme preparation. In this case, complete loss of PrP^(c)immunoreactivity is still apparent since no band is observable in lane3.

[0132] Preparation and Titration of Infectious Mouse Brain Homogenate

[0133] Eighty days post challenge onward, mice were subject to dailyclinical scoring to detect clinically affected mice as early aspossible. A single mouse died of an unknown cause prior to this period.The remainder exhibited the expected disease progression and weresacrificed between 110 and 130 days post challenge. The brains wereremoved aseptically and stored frozen until required. Forty-eight BSE(301V) infectious VM mouse brains were homogenised in four volumes ofPBS within a contained homogeniser then passed sequentially throughincreasingly fine gauge needles (21G to 26G) until free flowing. Asample with a further 2-fold dilution (1:9 mouse brain: PBS) wasprepared for titration of infectivity. Over 800×0.1 ml aliquots of BSE(301V) infectious mouse brain homogenate were prepared. These procedureswere again carried out under rigorous class Ill containment, includingthe wearing of positive pressure respirators.

[0134] Groups of 25 eight week old VM mice received titration doses ofthe infectious mouse brain homogenate preparation at 10 fold dilutionsfrom 10⁻¹-10⁻⁸. A further group of 25 mice were challenged withuninfectious mouse brain homogenate as a control. All mice wereinoculated under anaesthetic using 26G×⅜″ (0.95 cm) needles with plasticsleeve guards cut off 2 mm below bevel in a Class 2 cabinet with the useof an injection guard. The mice were then left to incubate the BSE(301V) agent for extended periods some in excess of a year. The initialtitre of the infectious mouse brain homogenate preparation wasestablished retrospectively once all incubations (clinical monitoring at80 days onwards) were complete.

[0135] Dimer Detection in Digested Mouse Brain

[0136] BSE (301V)—infected mouse brain homogenate was digested atneutral pH and 60° C. for 30 minutes with protease. Total proteindigests were run on SDS-PAGE and transferred by Western blotting tonitro-cellulose membranes. These were cut into strips and probed withCAMR anti-prion antibodies (produced in rabbits). A second genericantibody (goat anti-rabbit) was conjugated to horseradish peroxidase andused with detection by TMB calorimetric substrate.

[0137] At the time, the expected result was that the results were thesame as the control blot (number 7).—using the anti-prion antibody mAb6H4 (from Prionics, Switzerland). In this control blot, there is seenthe typical three-banded pattern (glycosylation states) forprotease-digested infectious-conformation prion protein (PrP^(Sc)).

[0138] However, the blots in this example did not show this pattern.Blot 1 uses a polyclonal antibody raised against a PPD-conjugatedpeptide corresponding to an N-terminal region of the prion molecule.Nothing is seen in the lanes. This section of the protein is susceptibleto proteolysis, so it is not surprising to see nothing in the lanes (2 &3)—see FIG. 9, blot 1 on left hand side. Lane 1 is a molecular weightmarker.

[0139] Blot 2 has a second antibody raised against a peptide sequencefurther into the prion molecule. This shows at least 9 bands of varyingintensity, approximately equidistant, at a molecular weightcorresponding to a prion dimer with a range of glycosylation states—seeblot 2 on FIG. 9.

[0140] Blot 3 antibody shows similar profile; blot 4 is also shown butits results are too poor quality to draw any conclusions—see blots 3 and4 on FIG. 9.

[0141] Blots 5 and 6, shown on FIG. 10 with the control blot 7, againshow the multibanded pattern

[0142] Dimer Detection in Digested Mouse Brain

[0143] The above example was repeated, and the results shown in FIGS. 11and 12.

[0144] Blot 1 shows molecular weight markers in lanes 1 and 5. Lane 2 isrecombinant murine PrP showing recombinant murine PrP oligomers. Lane 3shows lack of antibody response to protease-digested infectious mousebrain homogenate. Lane 4 is the antibody response in the undigestedcontrol.

[0145] Blot 2 is as above but shows the previous banding pattern in theprotease digested sample.

[0146] Blot 3 shows the antibody 3 response. Here there is some responseto recombinant murine PrP (lane 2). Lane 3 shows not only the multiple(dimeric PrP) banding pattern, but also some monomeric PrP response.

[0147] Blot 7 is the 6H4 mAb antibody control. Here there is gooddetection of recombinant murine PrP oligomers (lane 2). Lane 3 shows theheavily diglycosylated form of limitedly protease-treated PrP^(Sc), plusthe more minor monoglycosylated and non-glycosylated forms typical ofBSE (301V) strain. No ‘dimer’ detection is apparent.

[0148] Preparation of Antibodies Including Dimer Preferential Antibody

[0149] In the examples, we have used 6 polyclonal antibodies Of these,three detect the dimer alone and do not bind the monomer whereas onecross-reacts with both the monomer and the dimer.

[0150] The polyclonal sera were produced by immunisation of rabbits withsynthesised prion mimetic peptides. These peptides were designed basedon regions of high homology between human, mouse and bovine prionprotein amino acid sequences.

[0151] The sequences producing the dimer-reactive antibodies were asfollows: CGGWGQPHGGC (Peptide 2) CGGYMLGSAMSRPIIHFGNDYEC (Peptide 3)CVNITIKQHTVTTTTKGENFTETDC (Peptide 5) CITQYQRESQAYYQRGASC (Peptide 6)

[0152] The peptides were synthesised with a cysteine at both ends (seeabove) and with a cysteine at one end only. This method was used inorder to present both the linear form and a loop structure of theantigen on the surface of the carrier protein.

[0153] The peptides were synthesised commercially and coupled to thecarrier protein PPD (purified protein derivative), derived from anattenuated strain of the bacterium Mycobacterium bovis, which islyophilised and used to conjugate to the peptide via a linker.

[0154] Anti-prion polyclonal antibodies were produced as follows:

[0155] A sample of pre-immune sera (˜1 ml) was collected from each of agroup of Dutch rabbits.

[0156] The rabbits were injected with reconstituted freeze-driedBacillus Calmette-Guerin (BCG) vaccine for intradermal use. A dose of0.1 ml of reconstituted BCG vaccine was given in two sites in the scruffof the neck of the rabbit.

[0157] After 4 weeks, 0.6 mg of each peptide-PPD conjugate was measured(0.3 mg of each of the 1 cysteine and 2 cysteine versions) and dissolvedin 1 ml of sterile 0.9% saline.

[0158] An equal volume of incomplete Freunds adjuvant was added and 0.75ml aliquots, of the resulting emulsion, were injected intra-muscularlyinto each hind limb and 0.25 ml aliquots into two sites in the scruff ofthe neck per rabbit.

[0159] After 4 weeks a boost injection was given comprising of thepeptide-PPD conjugates prepared as in step 3 and 4. The boost injectionsconsist of four 0.25 ml injections into the scruff of the neck of eachrabbit.

[0160] 7-14 days after the first boost injections, 4 ml test bleeds weretaken, the sera was assessed by ELISA for antibody titre.

[0161] A second boost injection was given 4-6 weeks after the first.

[0162] A third boost injection given 4-6 weeks later.

[0163] A 4 ml test bleed was taken 6-8 weeks after the third boostinjection and antibody titres determined by ELISA

[0164] A fourth boost injection given.

[0165] A 4 ml test bleed was taken 7-14 days after the fourth boostinjection and antibody titre determined by ELISA.

[0166] Terminal exsanguination was carried out and blood collected. Theserum was separated by centrifugation and stored at −20° C.

[0167] Analysis of antibody titre was achieved using ELISA. Theimmunoassay plate was coated with the same peptides conjugated to adifferent carrier protein (KLH) in order to differentiate the responseto the peptide from the response to the carrier protein.

[0168] Three of the antibodies produced by immunisation of the syntheticpeptide sequences described bind preferentially to the dimer form of themolecule.

[0169] Analagous steps may also be used to prepare a monoclonalantibody. This could be achieved using a method such as described inAntibodies—A Laboratory Manual, Ed Harlow and David Lane, 1988 (ColdSpring Harbor Laboratory).

Example 2

[0170] Evaluation of Proteases MC-A, MC-3 and MC-4

[0171] Three new proteases, MC-A, MC-3 and MC-4, were assessed usinginfectious BSE (301V) mouse brain homogenate (mbh) dialysed to pH2,4,6,8,10 and 12 respectively and digested at 50° C. for 30 minutes.Total protein digests were run on SDS-PAGE and transferred by Westernblotting to nitro-cellulose membranes.

[0172] The Western blots were detected with 6H4 and TMB colorimetricsubstrate, and the results shown in FIG. 16.

[0173]FIG. 16 demonstrates characteristic PrP^(Sc) monomer bands presentafter digestion with the new proteases at pH 2-10. The monomer bandspresent after digestion with MC-3 at pH 2-10 appear very faint. Indeed,of the three proteases tested, MC-3 shows the greater reduction ofmonomer.

[0174] In addition, no PrP^(Sc) monomer bands are present at pH 12 withany of the three proteases.

[0175] Comparison of New Proteases with Properase

[0176] The digestion of PrP^(Sc) monomers using the three new proteaseswas compared to digestion using Properase, and the results shown inFIGS. 17 and 18.

[0177]FIG. 17 demonstrates monomer digestion at pH 2-12 by MC-A, MC-3and MC-4 at a temperature of 50° C., compared to Properase at 60° C. At50° C., the three new proteases give improved monomer digestion comparedto Properase at 60° C.

[0178]FIG. 18 shows monomer digestion at pH 2-12 by MC-A, MC-3 and MC-4at a temperature of 60° C., compared to Properase at 60° C. At 60° C.,digestion of the monomers with the three new proteases is comparable tothat using Properase.

[0179] Temperature Profiling of MC-3

[0180] Th effect of temperature on monomer digestion by MC-3 wasinvestigated, with results shown in FIG. 19.

[0181]FIG. 19 shows increased temperature to result in increasinglyincomplete digestion of mbh at low pH. However, at high pH (pH 10 andabove), increased temperature leads to less distinct monomer bands. Thissuggests that at increased temperatures, high pH enhances digestion ofmonomer by MC-3. It is also noted that MC-3 at 50° C. has a greatereffect across the pH range than either MC-A or MC-4.

[0182] Detection with PAb2

[0183] Mouse brain homogenates at pH 2-12 were digested at 50° C. for 30minutes and the corresponding Western blots detected with achemiluminescent detection substrate, PAb2. The results are shown inFIG. 20.

[0184]FIG. 20 shows high molecular weight (HMW) bands present afterdigestion with each protease at pH 2-10. The HMW bands at pH 12 are muchreduced with all three proteases. This implies that the HMW bands areproteinaceous, as they can be removed by proteases. These bands arethought to represent PrP^(Sc) dimers.

[0185] MC-3 Dilutions at pH 10 and pH 12

[0186]FIG. 21 shows the results of MC-3 dilutions at pH 10 and pH 12.

[0187] At pH 10, monomer bands are seen at 1:20 dilution and HMW bandsare seen across the dilution range. However, at pH 12, no monomer bandsare apparent and HMW bands are much reduced across the dilution range.This suggests high pH enables MC-3 to digest HMW dimer.

[0188] Comparison with Proteinase K

[0189] The ability of the three new proteases to digest monomer and HMWdimer was compared to Proteinase K, and the results shown in FIG. 22.

[0190] The results indicate that all three new proteases are better atremoving both the monomer and the HMW dimer than Proteinase K.

[0191] Summary of Results

[0192] Of the proteases tested, MC-3 shows the greater reduction of themonomers.

[0193] MC-3 is better at removing the monomer bands across the pH rangethan either Properase or Proteinase K. Unlike Properase or Proteinase K,the new proteases reduce the HMW bands at pH 12.

Example 3

[0194] Protocol for Protease Digestion of Mouse Brain Homogenate (MBH).

[0195] BSE (301V) infectious VM mouse brain homogenate was digested withMC3, MC4, proteinase K, properase, Purafect or Purafect ox and used toinfect VM mice as outlined below:

[0196] Preparation of Protease Treated Infectious MBH and Controls.

[0197] 2×500 μl of infectious MBH was microdialysed against an excess ofpH1 2 buffer (or as appropriate) for 30 minutes. The aliquots werecombined and separated into 2 lots:

[0198] Lot 1: 100 μl of infectious MBH as positive control

[0199] Transfer 90 μl to fresh tube and add 10 ml of H2O

[0200] Heat at 50° C. for 30 minutes (or as appropriate)

[0201] Neutralise by addition of 11 μl of 10×phosphate buffer, pH7.0

[0202] Heat at 100° C. for 10 minutes

[0203] Dilute with 889 μl of PBS

[0204] Mix, take 100 μl aliquot and dilute in further 900 μl PBS(overall 1:100 dilution)

[0205] 1 ml positive control ready for inoculation

[0206] Lot 2: 900 μl of infectious MBH for protease treatment

[0207] Divide into 10×90 μl aliquots

[0208] Add 10 μl of protease solution (neat) to each aliquot

[0209] Heat at 50° C. for 30 minutes (or as appropriate)

[0210] Neutralise by addition of 11 μl of 10×phosphate buffer, pH7.0

[0211] Heat at 100° C. for 10 minutes

[0212] Pool all aliquots and mix

[0213] 1 ml of infectivity test material ready for inoculation

[0214] Preparation of Controls to Assess Toxicity of Non-Infectious MBHin the Presence or Absence of Protease.

[0215] Additional controls to assess the toxicity of protease treatedMBH (in the absence of infectivity) were prepared as described below:

[0216] Microdialyse 1×500 μl of uninfectious MBH against pH 12 buffer(or as appropriate)

[0217] Divide into 5×90 μl aliquots

[0218] Add 10 ml of protease solution (neat) to each aliquot

[0219] Heat at 50° C. for 30 minutes (or as appropriate)

[0220] Neutralise by addition of 11 μl of 10×phosphate buffer, pH7.0

[0221] Heat at 100° C. for 10 minutes

[0222] Pool all aliquots and mix

[0223] 0.5 ml of toxicity test material ready for inoculation

[0224] Inoculation of VM Mice with Mouse Brain Homogenate.

[0225] VM mice were inoculated with 20 μl test material intracerebrallyaccording to published methods. The test samples were MC3, MC4,proteinase K, properase, Purafect and Purafect ox digested infectiousMBH, infectious MBH treated at pH 12 in the absence of protease, andtoxicity controls of protease treated MBH (in the absence ofinfectivity) and the titration series.

[0226] Mice were scored on the basis of clinical symptoms and sacrificedat a defined clinical end-point. The results are shown below andexpressed as the mean incubation period before sacrifice TABLE 1 Meanassuming Number of any remaining healthy Last death mice are all mice;no Number First (number of sacrificed on clinical Treatment of micedeath survivors) current day SD symptoms MBH study MC3 18 173 >277 (13)259.16 31.56 10 MC4 20 143 147 145.95 1.41 Proteinase 22 187 >282 (11)254.9 35.66  0 K Properase 24 137 169 147.54 7.71 Purafect 25 112 144132.04 8.23 Purafect Ox 24 125 146 132.83 4.76 Titration study;infectious MBH (iMBH) Positive 15 133 167 142.87 9.62 control 1:100 MBH× 1 10 112 142 120 8.5 MBH × 10⁻¹ 16 117 142 125.37 7.16 MBH × 10⁻² 23124 143 135.78 5.59 MBH × 10⁻³ 23 126 168 141.57 11.04 MBH × 10⁻⁴ 25 137198 157.52 18.5 MBH × 10⁻⁵ 25 150 448 226.96 94.81 # properase and theB. licheniformis subtilisin MC4 reduce the levels of infectivity bygreater than 3-logs (mean incubation times of 147.54 and 145.95 dayscompared to an incubation at 10⁻³ dilution of 141.57 days). MC3 andproteinase K both reduce the levels of infectivity by significantly morethan 5 logs and exceed the lowest detection levels of the assay with anumber of mice remaining alive after the incubation shown # (277 and 282days respectively). Of the 2 enzymes, treatment with MC3 shows thepresence of >50% of mice with no clinical symptoms at 277 days whilstthose surviving after treatment of infectious material with proteinase Kall show some signs of clinical disease after 282 days. Treatment witheither Purafect or Purafect Ox reduce the levels of infectivity bynearly 2 logs whilst pH treatment alone results in a 1 log reduction ininfectivity.

Example 4

[0227] Protocol for Protease Digestion of Meat and Bone Meal (MBM).

[0228] BSE (301V) infectious VM mouse brain homogenate was spiked into abackground of meat and bone meal (MBM), digested with MC3, MC4 and usedto infect VM mice as outlined below.

[0229] The method is designed to assess the ability of the proteases toinactivate TSEs in a protein-rich background. Such conditions areidentical to those that would be encountered in meat rendering processeswhere the presence of TSE material in meat waste would be eliminated bytreatment with protease. The results therefore demonstrate that themethod of the invention is suitable for the large scale inactivation ofTSE agents as a precursor to meat rendering, for the decontamination ofinfected meat waste or for other processes where inactivation of TSEs isrequired prior to further applications.

[0230] Preparation of Protease-Treated Infectious-MBH Spiked MBM andControls

[0231] To evaluate the ability of proteases to inactivate infectivematerial in a background of MBM samples were prepared and treated asfollows:

[0232] 2×100 mg aliquots of MBM were prepared in tubes

[0233] Add 700 μl of pH 12 buffer to each tube

[0234] Add 100 μl of infectious MBH dialysed to pH12 to each tube

[0235] Add 100 μl of protease solution (neat) to each tube

[0236] Heat at 60° C. for 30 minutes

[0237] Neutralise by addition of 100 μl of 10×phosphate buffer to eachtube

[0238] Heat at 100° C. for 10 minutes

[0239] Allow samples to settle and draw off supernatant

[0240] Pool supernatant and mix, check pH is ˜7.0 ˜2 ml of infectivitytest material ready for inoculation

[0241] Preparation of Positive Control Sample Incorporating ProteaseTreated MBM Spiked with Untreated Infectious.

[0242] Positive controls were prepared in the presence of proteasetreated MBM to ensure that no toxic effects were observed as acombination of digested MBM and infectious material. Samples wereprepared and treated as described below:

[0243] Digestion 1

[0244]4×100 mg aliquot of MBM in tubes

[0245] Add 700 μl of pH 12 buffer to each tube

[0246] Add 100 μl of protease solution (neat) to each tube

[0247] Heat at 60° C. for 30 minutes

[0248] Neutralise by addition of 90 μl of 10×phosphate buffer each tube

[0249] 1. Heat at 100° C. for 10 minutes

[0250] 2. Allow sample to settle and draw off supernatant (900 μl)

[0251] Digestion 2

[0252] 100 μl of infectious MBH dialysed to pH12

[0253] Heat at 60° C. for 30 minutes

[0254] Neutralise by addition of 10 μl of 10×phosphate buffer

[0255] Heat at 100° C. for 10 minutes

[0256] Dilute sample with 890 μl PBS (1:10)

[0257] Take 100 μl of this sample and add 900 μl of supernatant fromdigestion 1 (1:100)

[0258] Check pH is ˜7.0, ˜1 ml of positive test material ready forinoculation

[0259] Preparation of Material to Assess Toxicity of Protease TreatedMBM Alone

[0260] The toxicity of protease treated MBM in the absence ofinfectivity was assessed using MBM spiked with non-infectious MBH priorto treatment. Samples were prepared as described below:

[0261] 1×100 mg aliquot of MBM in tube

[0262] Add 700 μl of pH 12 buffer

[0263] Add 100 μl of non-infectious MBH dialysed to pH12

[0264] Add 100 μl of protease solution (neat)

[0265] Heat at 60° C. for 30 minutes

[0266] Neutralise by addition of 100 μl of 10×phosphate buffer

[0267] Heat at 100° C. for 10 minutes

[0268] Allow sample to settle and draw off supernatant, check pH is ˜7.0

[0269] ˜1 ml of toxicity/negative test material ready for inoculation

[0270] Inoculation of VM Mice with Mouse Brain Homogenate.

[0271] VM mice were inoculated with 20 μl test material intracerebrallyaccording to published methods. The test samples were MC3, MC4 andproteinase K treated infectious MBH in MBM (test groups), infectious MBHtreated at pH 12, mixed with protease-treated MBM (positive controlgroups for each protease) and protease-treated non-infectious MBH in MBM(negative control). TABLE 2 Mean assuming any Number of remaininghealthy Last death mice are all mice; no No. of (number of sacrificed onclinical Treatment mice First death survivors) current day SD symptomsMBM study MC3 positive 15 153 >192 (1) 161.13 12.2  0 MC3 25 >192(24) >192 12 MC4 positive 15 143 158 148.13 4.36 MC4 24 >186 (25) >18625 # days for MC3 and >38 days for MC4 both equate to greater than 4 logreduction in infectivity based on the MBH titration study described inthe previous example.

[0272] The invention thus provides for the detection and degradation ofTSE infectivity. TABLE 3 Organism Domain Growth T opt pH opt Aeropyrumpernix Archaeon Aerobe 95° C. 7.0 Alicyclobacillus acidocaldariusBacterium Aerobe 65° C. 3.5 Archaeoglobus fulgidus Archaeon Anaerobe 85°C. 6.5 Bacillus caldotenax BTI Bacterium Aerobe 65° C. 7 Bacilluspallidus Bacterium Aerobe 65° C. 9.0 Bacillus stearothermophilus L32-65Bacterium Aerobe 65° C. 7.0 Bacillus stearothermophilus LUDA T57Bacterium Aerobe 65° C. 7.0 Bacillus thermoproteolyticus Rokko BacteriumAerobe 65° C. 7.0 Bacillus sp. 11231 Bacterium Aerobe 65° C. 7.0Bacillus sp. 11276 Bacterium Aerobe 65° C. 7 Bacillus sp. 11652Bacterium Aerobe 65° C. 7 Bacillus sp. 12031 Bacterium Aerobe 65° C. 7Desulfurococcus sp. Archaeon Anaerobe 85° C. 6.5 Fervidobacteriumpennivorans Bacterium Anaerobe 70° C. 8.5 Hyperthermus butylicusArchaeon Anaerobe 95° C. 6.5 Pyrococcus furiosus Archaeon Anaerobe 95°C. 75 Pyrococcus horikoshii Archaeon Anaerobe 95° C. 7 Sulfolobusacidocaldarius 98-3 Archaeon Aerobe 75° C. 2.5 Sulfolobus hakonensisArchaeon Aerobe 75° C. 2.5 Sulfolobus solfataricus P1 Archaeon Aerobe75° C. 2.5 Sulfolobus solfataricus P2 Archaeon Aerobe 75° C. 2.5Thermobrachium celere Bacterium Anaerobe 65° C. 8.5 Thermococcusfumicolans Archaeon Anaerobe 85° C. 6.5 Thermus caldophilus GK24Bacterium Aerobe 70° C. 8.0 Thermus aquaticus YT1 Bacterium Aerobe 70°C. 8.0 Thermus sp. 16132 Bacterium Aerobe 70° C. 8.0 Thermus sp. 15673Bacterium Aerobe 70° C. 8.0 Thermus sp. Rt41A Bacterium Aerobe 70° C. 8

[0273]

1 12 1 11 PRT Artificial Sequence Synthetic 1 Cys Gly Gly Trp Gly GlnPro His Gly Gly Cys 1 5 10 2 23 PRT Artificial Sequence Synthetic 2 CysGly Gly Tyr Met Leu Gly Ser Ala Met Ser Arg Pro Ile Ile His 1 5 10 15Phe Gly Asn Asp Tyr Glu Cys 20 3 25 PRT Artificial Sequence Synthetic 3Cys Val Asn Ile Thr Ile Lys Gln His Thr Val Thr Thr Thr Thr Lys 1 5 1015 Gly Glu Asn Phe Thr Glu Thr Asp Cys 20 25 4 19 PRT ArtificialSequence Synthetic 4 Cys Ile Thr Gln Tyr Gln Arg Glu Ser Gln Ala Tyr TyrGln Arg Gly 1 5 10 15 Ala Ser Cys 5 1497 DNA Bacillus amyloliquefaciens5 ggtctactaa aatattattc catactatac aattaataca cagaataatc tgtctattgg 60ttattctgca aatgaaaaaa aggagaggat aaagagtgag aggcaaaaaa gtatggatca 120gtttgctgtt tgctttagcg ttaatcttta cgatggcgtt cggcagcaca tcctctgccc 180aggcggcagg gaaatcaaac ggggaaaaga aatatattgt cgggtttaaa cagacaatga 240gcacgatgag cgccgctaag aagaaagatg tcatttctga aaaaggcggg aaagtgcaaa 300agcaattcaa atatgtagac gcagcttcag tcacattaaa cgaaaaagct gtaaaagaat 360tgaaaaaaga cccgagcgtc gcttacgttg aagaagatca cgtagcacat gcgtacgcgc 420agtccgtgcc ttacggcgta tcacaaatta aagcccctgc tctgcactct caaggctaca 480ctggatcaaa tgttaaagta gcggttatcg acagcggtat cgattcttct catcctgatt 540taaaggtagc aagcggagcc agcatggttc cttctgaaac aaatcctttc caagacaaca 600actctcacgg aactcacgtt gccggcacag ttgcggctct taataactca atcggtgtat 660taggcgttgc gccaagcgca tcactttacg ctgtaaaagt tctcggtgct gacggttccg 720gccaatacag ctggatcatt aacggaatcg agtgggcgat cgcaaacaat atggacgtta 780ttaacatgag cctcggcgga ccttctggtt ctgctgcttt aaaagcggca gttgataaag 840ccgttgcatc cggcgtcgta gtcgttgcgg cagccggtaa cgaaggcact tccggcagct 900caagcacagt gggctaccct ggtaaatacc cttctgtcat tgcagtaggc gctgttgaca 960gcagcaacca aagagcatct ttctcaagcg taggacctga gcttgatgtc atggcacctg 1020gcgtatctat ccaaagcacg cttcctggaa acaaatacgg ggcgtacaac ggtacgtcaa 1080tggcatctcc gcacgttgcc ggagcggctg ctttgattct ttctaagcac ccgaactgga 1140caaacactca agtccgcagc agtttagaaa acaccactac aaaacttggt gattctttgt 1200actatggaaa agggctgatc aacgtacaag cggcagctca gtaaaacata aaaaaccggc 1260cttggccccg ccggtttttt attatttttc ttcctccgca tgttcaatcc gctccataat 1320cgacggatgg ctccctctga aaattttaac gagaaacggc gggttgaccc ggctcagtcc 1380cgtaacggcc aactcctgaa acgtctcaat cgccgcttcc cggtttccgg tcagctcaat 1440gccataacgg tcggcggcgt tttcctgata ccgggagacg gcattcgtaa tcggatc 1497 6382 PRT Bacillus amyloliquefaciens 6 Met Arg Gly Lys Lys Val Trp Ile SerLeu Leu Phe Ala Leu Ala Leu 1 5 10 15 Ile Phe Thr Met Ala Phe Gly SerThr Ser Ser Ala Gln Ala Ala Gly 20 25 30 Lys Ser Asn Gly Glu Lys Lys TyrIle Val Gly Phe Lys Gln Thr Met 35 40 45 Ser Thr Met Ser Ala Ala Lys LysLys Asp Val Ile Ser Glu Lys Gly 50 55 60 Gly Lys Val Gln Lys Gln Phe LysTyr Val Asp Ala Ala Ser Val Thr 65 70 75 80 Leu Asn Glu Lys Ala Val LysGlu Leu Lys Lys Asp Pro Ser Val Ala 85 90 95 Tyr Val Glu Glu Asp His ValAla His Ala Tyr Ala Gln Ser Val Pro 100 105 110 Tyr Gly Val Ser Gln IleLys Ala Pro Ala Leu His Ser Gln Gly Tyr 115 120 125 Thr Gly Ser Asn ValLys Val Ala Val Ile Asp Ser Gly Ile Asp Ser 130 135 140 Ser His Pro AspLeu Lys Val Ala Ser Gly Ala Ser Met Val Pro Ser 145 150 155 160 Glu ThrAsn Pro Phe Gln Asp Asn Asn Ser His Gly Thr His Val Ala 165 170 175 GlyThr Val Ala Ala Leu Asn Asn Ser Ile Gly Val Leu Gly Val Ala 180 185 190Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu Gly Ala Asp Gly Ser 195 200205 Gly Gln Tyr Ser Trp Ile Ile Asn Gly Ile Glu Trp Ala Ile Ala Asn 210215 220 Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly Pro Ser Gly Ser Ala225 230 235 240 Ala Leu Lys Ala Ala Val Asp Lys Ala Val Ala Ser Gly ValVal Val 245 250 255 Val Ala Ala Ala Gly Asn Glu Gly Thr Ser Gly Ser SerSer Thr Val 260 265 270 Gly Tyr Pro Gly Lys Tyr Pro Ser Val Ile Ala ValGly Ala Val Asp 275 280 285 Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser ValGly Pro Glu Leu Asp 290 295 300 Val Met Ala Pro Gly Val Ser Ile Gln SerThr Leu Pro Gly Asn Lys 305 310 315 320 Tyr Gly Ala Tyr Asn Gly Thr SerMet Ala Ser Pro His Val Ala Gly 325 330 335 Ala Ala Ala Leu Ile Leu SerLys His Pro Asn Trp Thr Asn Thr Gln 340 345 350 Val Arg Ser Ser Leu GluAsn Thr Thr Thr Lys Leu Gly Asp Ser Leu 355 360 365 Tyr Tyr Gly Lys GlyLeu Ile Asn Val Gln Ala Ala Ala Gln 370 375 380 7 275 PRT Bacillusamyloliquefaciens 7 Ala Gln Ser Val Pro Tyr Gly Val Ser Gln Ile Lys AlaPro Ala Leu 1 5 10 15 His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys ValAla Val Ile Asp 20 25 30 Ser Gly Ile Asp Ser Ser His Pro Asp Leu Lys ValAla Gly Gly Ala 35 40 45 Ser Met Val Pro Ser Glu Thr Asn Pro Phe Gln AspAsn Asn Ser His 50 55 60 Gly Thr His Val Ala Gly Thr Val Ala Ala Leu AsnAsn Ser Ile Gly 65 70 75 80 Val Leu Gly Val Ala Pro Ser Ala Ser Leu TyrAla Val Lys Val Leu 85 90 95 Gly Ala Asp Gly Ser Gly Gln Tyr Ser Trp IleIle Asn Gly Ile Glu 100 105 110 Trp Ala Ile Ala Asn Asn Met Asp Val IleAsn Met Ser Leu Gly Gly 115 120 125 Pro Ser Gly Ser Ala Ala Leu Lys AlaAla Val Asp Lys Ala Val Ala 130 135 140 Ser Gly Val Val Val Val Ala AlaAla Gly Asn Glu Gly Thr Ser Gly 145 150 155 160 Ser Ser Ser Thr Val GlyTyr Pro Gly Lys Tyr Pro Ser Val Ile Ala 165 170 175 Val Gly Ala Val AspSer Ser Asn Gln Arg Ala Ser Phe Ser Ser Val 180 185 190 Gly Pro Glu LeuAsp Val Met Ala Pro Gly Val Ser Ile Gln Ser Thr 195 200 205 Leu Pro GlyAsn Lys Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Ser 210 215 220 Pro HisVal Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn 225 230 235 240Trp Thr Asn Thr Gln Val Arg Ser Ser Leu Gln Asn Thr Thr Thr Lys 245 250255 Leu Gly Asp Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala 260265 270 Ala Ala Gln 275 8 275 PRT Bacillus subtilis 8 Ala Gln Ser ValPro Tyr Gly Ile Ser Gln Ile Lys Ala Pro Ala Leu 1 5 10 15 His Ser GlnGly Tyr Thr Gly Ser Asn Val Lys Val Ala Val Ile Asp 20 25 30 Ser Gly IleAsp Ser Ser His Pro Asp Leu Asn Val Arg Gly Gly Ala 35 40 45 Ser Phe ValPro Ser Glu Thr Asn Pro Tyr Gln Asp Gly Ser Ser His 50 55 60 Gly Thr HisVal Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly 65 70 75 80 Val LeuGly Val Ser Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu 85 90 95 Asp SerThr Gly Ser Gly Gln Tyr Ser Trp Ile Ile Asn Gly Ile Glu 100 105 110 TrpAla Ile Ser Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly 115 120 125Pro Thr Gly Ser Thr Ala Leu Lys Thr Val Val Asp Lys Ala Val Ser 130 135140 Ser Gly Ile Val Val Ala Ala Ala Ala Gly Asn Glu Gly Ser Ser Gly 145150 155 160 Ser Thr Ser Thr Val Gly Tyr Pro Ala Lys Tyr Pro Ser Thr IleAla 165 170 175 Val Gly Ala Val Asn Ser Ser Asn Gln Arg Ala Ser Phe SerSer Ala 180 185 190 Gly Ser Glu Leu Asp Val Met Ala Pro Gly Val Ser IleGln Ser Thr 195 200 205 Leu Pro Gly Gly Thr Tyr Gly Ala Tyr Asn Gly ThrSer Met Ala Thr 210 215 220 Pro His Val Ala Gly Ala Ala Ala Leu Ile LeuSer Lys His Pro Thr 225 230 235 240 Trp Thr Asn Ala Gln Val Arg Asp ArgLeu Glu Ser Thr Ala Thr Tyr 245 250 255 Leu Gly Asn Ser Phe Tyr Tyr GlyLys Gly Leu Ile Asn Val Gln Ala 260 265 270 Ala Ala Gln 275 9 274 PRTBacillus licheniformis 9 Ala Gln Thr Val Pro Tyr Gly Ile Pro Leu Ile LysAla Asp Lys Val 1 5 10 15 Gln Ala Gln Gly Phe Lys Gly Ala Asn Val LysVal Ala Val Leu Asp 20 25 30 Thr Gly Ile Gln Ala Ser His Pro Asp Leu AsnVal Val Gly Gly Ala 35 40 45 Ser Phe Val Ala Gly Glu Ala Tyr Asn Thr AspGly Asn Gly His Gly 50 55 60 Thr His Val Ala Gly Thr Val Ala Ala Leu AspAsn Thr Thr Gly Val 65 70 75 80 Leu Gly Val Ala Pro Ser Val Ser Leu TyrAla Val Lys Val Leu Asn 85 90 95 Ser Ser Gly Ser Gly Ser Tyr Ser Gly IleVal Ser Gly Ile Glu Trp 100 105 110 Ala Thr Thr Asn Gly Met Asp Val IleAsn Met Ser Leu Gly Gly Ala 115 120 125 Ser Gly Ser Thr Ala Met Lys GlnAla Val Asp Asn Ala Tyr Ala Arg 130 135 140 Gly Val Val Val Val Ala AlaAla Gly Asn Ser Gly Asn Ser Gly Ser 145 150 155 160 Thr Asn Thr Ile GlyTyr Pro Ala Lys Tyr Asp Ser Val Ile Ala Val 165 170 175 Gly Ala Val AspSer Asn Ser Asn Arg Ala Ser Phe Ser Ser Val Gly 180 185 190 Ala Glu LeuGlu Val Met Ala Pro Gly Ala Gly Val Tyr Ser Thr Tyr 195 200 205 Pro ThrAsn Thr Tyr Ala Thr Leu Asn Gly Thr Ser Met Ala Ser Pro 210 215 220 HisVal Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn Leu 225 230 235240 Ser Ala Ser Gln Val Arg Asn Arg Leu Ser Ser Thr Ala Thr Tyr Leu 245250 255 Gly Ser Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Glu Ala Ala260 265 270 Ala Gln 10 269 PRT Bacillus lentus 10 Ala Gln Ser Val ProTrp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala 1 5 10 15 His Asn Arg GlyLeu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp 20 25 30 Thr Gly Ile SerThr His Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser 35 40 45 Phe Val Pro GlyGlu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr 50 55 60 His Val Ala GlyThr Ile Ala Ala Leu Asn Asn Ser Ile Gly Val Leu 65 70 75 80 Gly Val AlaPro Ser Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala 85 90 95 Ser Gly SerGly Ser Val Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala 100 105 110 Gly AsnAsn Gly Met His Val Ala Asn Leu Ser Leu Gly Ser Pro Ser 115 120 125 ProSer Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly 130 135 140Val Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Gly Ser Ile Ser 145 150155 160 Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp Gln165 170 175 Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Ala Gly Leu AspIle 180 185 190 Val Ala Pro Gly Val Asn Val Gln Ser Thr Tyr Pro Gly SerThr Tyr 195 200 205 Ala Ser Leu Asn Gly Thr Ser Met Ala Thr Pro His ValAla Gly Ala 210 215 220 Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp SerAsn Val Gln Ile 225 230 235 240 Arg Asn His Leu Lys Asn Thr Ala Thr SerLeu Gly Ser Thr Asn Leu 245 250 255 Tyr Gly Ser Gly Leu Val Asn Ala GluAla Ala Thr Arg 260 265 11 275 PRT Artificial Sequence Bacillus subtilis11 Ala Gln Ser Val Pro Tyr Gly Ile Ser Gln Ile Lys Ala Pro Ala Leu 1 510 15 His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val Ala Val Ile Asp 2025 30 Ser Gly Ile Asp Ser Ser His Pro Asp Leu Asn Val Arg Gly Gly Ala 3540 45 Ser Phe Val Pro Ser Glu Thr Asn Pro Tyr Gln Asp Gly Ser Ser His 5055 60 Gly Thr His Val Ala Gly Thr Ile Ala Ala Leu Asp Asn Ser Ile Gly 6570 75 80 Val Leu Gly Val Ser Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu85 90 95 Asp Ser Thr Gly Ser Gly Ala Ile Ser Trp Ile Ile Asn Gly Ile Glu100 105 110 Trp Ala Ile Ser Asn Asn Met Asp Val Ile Asn Met Ser Leu GlyGly 115 120 125 Pro Thr Gly Ser Thr Ala Leu Lys Thr Val Val Asp Lys AlaVal Ser 130 135 140 Ser Gly Ile Val Val Ala Ala Ala Ala Gly Asn Glu GlySer Ser Gly 145 150 155 160 Ser Thr Ser Thr Val Gly Tyr Pro Ala Lys TyrPro Ser Thr Ile Ala 165 170 175 Val Gly Ala Val Asn Ser Ser Asn Gln ArgAla Ser Phe Ser Ser Ala 180 185 190 Gly Ser Glu Leu Asp Val Met Ala ProGly Val Ser Ile Gln Ser Thr 195 200 205 Leu Pro Gly Gly Thr Tyr Gly AlaTyr Asn Gly Thr Ser Met Ala Thr 210 215 220 Pro His Val Ala Gly Ala AlaAla Leu Ile Leu Ser Lys His Pro Thr 225 230 235 240 Trp Thr Asn Ala GlnVal Arg Asp Arg Leu Glu Ser Thr Ala Thr Tyr 245 250 255 Leu Gly Asn SerPhe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala 260 265 270 Ala Ala Gln275 12 269 PRT Artificial Sequence Bacillus lentus 12 Ala Gln Ser ValPro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala 1 5 10 15 His Asn ArgGly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp 20 25 30 Thr Gly IleSer Thr His Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser 35 40 45 Phe Val ProGly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr 50 55 60 His Val AlaGly Thr Ile Ala Ala Leu Asp Asn Ser Ile Gly Val Leu 65 70 75 80 Gly ValAla Pro Ser Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala 85 90 95 Ser GlySer Gly Ala Ile Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala 100 105 110 GlyAsn Asn Gly Met His Val Ala Asn Leu Ser Leu Gly Ser Pro Ser 115 120 125Pro Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly 130 135140 Val Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Gly Ser Ile Ser 145150 155 160 Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr AspGln 165 170 175 Asn Asn Asn Arg Ala Ser Pro Ser Gln Tyr Gly Ala Gly LeuAsp Ile 180 185 190 Val Ala Pro Gly Val Asn Val Gln Ser Thr Tyr Pro GlySer Thr Tyr 195 200 205 Ala Ser Leu Asn Gly Thr Ser Met Ala Thr Pro HisVal Ala Gly Ala 210 215 220 Ala Ala Leu Val Lys Gln Lys Asn Pro Ser TrpSer Asn Val Gln Ile 225 230 235 240 Arg Asn His Leu Lys Asn Thr Ala ThrSer Leu Gly Ser Thr Asn Leu 245 250 255 Tyr Gly Ser Gly Leu Val Asn AlaGlu Ala Ala Thr Arg 260 265

1. A method for inactivating a transmissible spongiform encephalopathy(TSE) agent comprising exposing the TSE agent to a thermostableproteolytic enzyme.
 2. The method of claim 1, comprising exposing theTSE agent to the thermostable protease at a temperature that is equal toor greater than 40° C.
 3. The method of claim 2, wherein the temperatureis between 50° C. and 120° C.
 4. The method of claim 3, wherein thetemperature is between 55° C. and 85° C.
 5. The method of claim 1,comprising exposing the TSE agent to the thermostable proteolytic enzymeat alkaline pH.
 6. The method of claim 5, wherein the pH is from 8 to13.
 7. The method of claim 5, wherein the pH is from 10 to
 12. 8. Themethod of claim 1 wherein the TSE agent is a prion.
 9. The method ofclaim 8, wherein the TSE agent is selected from the group consisting ofCreutzfeld-Jacob disease; variant Creutzfeld-Jacob disease; Kuru; fatalfamilial insomnia; Gerstmann-Straussler-Scheinker syndrome; bovinespongiform encephalopathy; scrapie; feline spongiform encephalopathy;chronic wasting disease; and transmissible mink encephalopathy.
 10. Themethod of claim 1, wherein the thermostable proteolytic enzyme isobtained from a thermophilic organism selected from the group consistingof archaea; hyperthermophilic bacteria and thermophilic bacteria. 11.The method of claim 10 wherein the thermophilic organism is selectedfrom the group consisting of Thermotoga maritima; Thermotoganeopolitana; Thermotoga thermarum; Fervidobacterium islandicum;Fervidobacterium nodosum; Fervidobacterium pennivorans; Thermosiphoafricanus; Aeropyrum pernix; Thermus flavus; pyrococcus spp.; Sulfolobussolfataricus; Desulfurococcus; Bacillus thermoproteolyticus; Bacillusstearo-thermophilus; Bacillus sp. 11231; Bacillus sp. 11276; Bacillussp. 11652; Bacillus sp. 12031; Thermus aquaticus; Thermus caldophilus;Thermus sp. 16132; Thermus sp. 15673; and Thermus sp. Rt41A.
 12. Amethod of sterilising apparatus comprising exposing said apparatus to asolution comprising a thermostable proteolytic enzyme.
 13. The method ofclaim 12, wherein the solution is maintained at a temperature below 100°C.
 14. The method of claim 12, wherein the solution is maintained at atemperature of between 45° C. and 85° C.
 15. The method of claim 12,wherein the solution has an alkaline pH.
 16. The method of claim 15,wherein the solution has a pH of between 8 and
 13. 17. The method ofclaim 12, wherein the thermostable proteolytic enzyme is obtained from athermophilic organism.
 18. The method of claim 17 wherein thethermophilic organism is selected from the group consisting of archaea;hyperthermophilic bacteria and thermophilic bacteria.
 19. The method ofclaim 12, wherein the solution is applied to the apparatus as a spray.20. The method of claim 12, wherein the apparatus is immersed in thesolution.
 21. A method of sterilising apparatus, comprising exposingsaid apparatus to a first solution comprising a first thermostableproteolytic enzyme; and exposing the apparatus to at least a secondsolution comprising a second thermostable proteolytic enzyme.
 22. Themethod of claim 21, wherein the first and second proteolytic enzymes arethe same.
 23. The method of claim 21, wherein the first proteolyticenzyme is different to the second proteolytic enzyme.
 24. The method ofclaim 21, wherein the pH of the first solution is different to the pH ofthe second solution.
 25. The method of claim 21, wherein the temperatureof the first solution is different to the temperature of the secondsolution.
 26. A composition for inactivating a TSE agent, comprising (1)a thermostable proteolytic enzyme and (2) a buffering agent having apK_(a) of from 8 to
 13. 27. The composition of claim 26, wherein thethermostable proteolytic enzyme is obtained from a thermophilic organismselected from the group consisting of archaea; hyperthermophilicbacteria and thermophilic bacteria.
 28. Apparatus for inactivating a TSEagent comprising: a. a chamber for receiving contaminated material; b.means for controlling the temperature of the chamber; and c. athermostable proteolytic enzyme active at alkaline pH, located withinthe chamber.
 29. A method of examining a sample infected with orsuspected to be infected by prion protein, comprising detecting dimersof prion protein in the sample.
 30. An antibody, which is specific forprion dimer but does not bind to prion monomer.
 31. The method of claim1, wherein the thermostable proteolytic enzyme is a serine protease. 32.The method of claim 1, wherein the thermostable proteolytic enzyme is asubtilisin.
 33. The method of claim 32, wherein the thermostableproteolytic enzyme is a subtilisin derived from Bacillus bacteria. 34.The method of claim 33 wherein the thermostable proteolytic enzyme is asubtilisin derived from Bacillus amyloliquefaciens, Bacillus lentus,Bacillus licheniformis, Bacillus subtilis or is subtilisin PB92.
 35. Themethod of claim 1, wherein the thermostable proteolytic enzyme isselected from the group consisting of MC-A, MC-3 and MC-4.